The detection and identification of bacterial pathogens in human or animal feces is essential for the diagnosis of certain diseases. The speed with which these diagnoses are made is important. Rapid disease diagnosis could be a reality if it were possible to detect bacterial pathogens directly in fecal matter using immunologically based or nucleic acid based probes. Unfortunately, however, feces (fecal matter) constitute a rather complex mixture of lipids (fats), cellulosic fibres, proteins, microorganisms and water; in certain animal fecal matter, it is not uncommon to find gravel, feathers and other impurities. As a result, fecal matter is an example of a very difficult-to-separate organic matter. Extraneous components in the fecal matter interfere with the detection of bacteria by most probes such as nucleic acid or antibody probes, and obviously by microscope.
No method is known to be available for the separation of bacteria from fecal matter for direct detection by probes. Fecal samples are usually cultured on selective media which support the growth of the bacteria. The growth can take 16 hours or longer, making a same-day diagnosis unfeasible. The culturing of the fecal matter dilutes the extraneous components which would normally interfere with the detection of the bacteria by probes or culture methods.
Attempts have been made to separate bacteria from the above-mentioned organic matter by filtration using a single filtering layer, or a number of identical filtering layers. These attempts were unsuccessful as the filtering layers clog very quickly with fecal, or similar, matter.
U.S. Pat. No. 3,668,925 issued Jun. 13, 1972 to Mesek describes a method of detecting the existence of leucocytes in milk by passing the milk through a first filter which retains gross impurities present in the milk and through a finer filter of higher density. The filters are then separated along an interface therebetween. A discoloration of the second filter is indicative of a high leucocyte count in the milk.
Leucocytes, of course, are 10-20 times larger than bacteria. Milk is a very different medium than feces and its filtration poses much less difficulties than of the latter.
The most relevant literature on the subject includes:
1) Cerqueria-Campos, M.-L., et al, Improved Immunological Membrane Filtration Methods for Detection of Food-born Salmonella Strains, Applied and Environmental Microbiology 52: 124-127, 1986. This paper describes a membrane filter method that involves the use of an enzyme-labelled antibody stain for the rapid detection of salmonella species in foods. The method allows detection of salmonella in foods within 48 hours.
2) Cox, N. A., et al, A Comparison of Various Enrichment Broths and Plating Media for the Isolation of Salmonella from Poultry Feces and Poultry Food Products, Poultry Science L1: 1312-1316, 1972.
This paper describes a study conducted to determine the efficacy of media prescribed by the FDA. A number of enrichment broths were tested. The experimental approach involved incubation, enrichment broth tubes were used to streak different plating media. The procedure takes more than 48 hours. There is a sizable percentage of false readings.
3) Flowers, R. S., Comparison of Rapid Salmonella Screening Methods and Conventional Culture Method, Outstanding symposia in Salmonella, 1984.
The author compares recently developed methods known as enzyme immunosorbent assays or enzyme immunoassays ElAs, and DNA-DNA hybridization assays with prior methods, fluorescent-antibody (FA) technique and Bacteriological Analytical Manual (BAM) culture procedure.
A common feature of all the above-mentioned methods, none of which is rapid enough to present reliable results in less time than ca. 16-24 hours, is that Salmonella is not physically separated from the solid matter, but rather enriched and subjected to a reaction with a specific detection agent.